Algicidal activity and gliding motility of Saprospira sp. SS98-5

A marine bacterium, Saprospira sp. SS98-5, which was isolated from Kagoshima Bay, Japan, was able to kill and lyse the cells of the diatom Chaetoceros ceratosporum. The multicellular filamentous cells of this bacterium captured the diatom cells, formed cell aggregates, and lysed them in an enriched sea water (ESS) liquid medium. Strain SS98-5 also formed plaques on double layer agar plates incorporating diatom cells. The diatom cell walls were partially degraded at the contact sites with the bacteria, the bacteria invaded from there into the diatom cells, and then the diatom cells were completely lysed. The strain possessed gliding motility and grew as spreading colonies on ESS agar plates containing lower concentrations of polypeptone (below 0.1%) while forming nonspreading colonies on ESS agar plates containing 0.5% polypeptone. Electron micrographs of ultrathin sections demonstrated that microtubule-like structures were observable only in gliding motile cells. Both the gliding motility and the microtubule-like structures were diminished by the addition of podophyllotoxin, an inhibitor of microtubule assembly, suggesting that the microtubule-like structures observed in these bacterial cells are related to their gliding motility.     

溶原性噬菌体在猪链球菌2型分离株中的检出和鉴定

[目的]为了研究噬菌体整合酶基因在猪链球菌2型(Streptococcus suis type 2,SS2)中的分布情况.[方法]根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序.[结果]结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段.经丝裂霉素C诱导后,SS2致病菌株出现完全的细胞溶解,而非毒力株T15未出现溶解.SS2致病株HA9801和ZY05719诱导均产生溶原性噬菌体,分别命名为SS2-HA和SS2-ZY,电镜观察,二者均头部呈正六边形,无尾部,其核酸类型为dsDNA,可鉴定为复层噬菌体科(Tectiviridae)的成员.噬菌体SS2-HA和SS2-ZY整合酶基因序列与已报道的SS2噬菌体整合酶基因序列高度同源,显示SS2噬菌体整合酶具有较高的特异性.[结论]从SS2致病株中检出溶原性噬菌体和噬菌体整合酶基因,且噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关.     

采用苯酚羟化酶基因特异引物检测苯酚降解菌

根据苯酚羟化酶基因高度保守序列设计了一对该基因的特异PCR引物。采用该特异引物从苯酚降解菌醋酸钙不动杆菌 (Acinetobactercalcoaceticus)PHEA 2的总DNA中扩增到唯一一条大小为 684bp的片段。该DNA片段与已知的A .calcoaceticusNCIB82 50的苯酚羟化酶基因具有高度的同源性 ,其核苷酸序列的同源性为 84% ,推导的氨基酸序列的同源性为 98%。对苯酚和非苯酚降解菌株的PCR扩增结果表明 :所有苯酚降解菌均能扩增出 684bp的特征片段 ,而非苯酚降解菌则无PCR条带。对炼焦废水中的细菌群落进行PCR扩增和生化特性检测表明 :显示 684bp特征片段的菌株均具有苯酚降解特性。上述结果表明 ,利用苯酚羟化酶基因的特异引物可对环境中的苯酚降解菌株进行准确快速的PCR检测。     

Specifics of the coat protein gene in Russian strains of the cucumber green mottle mosaic virus

The primary structure of the coat protein (CP) gene was examined for pathogenic strain MS-1 and vaccine strain VIROG-43M of the cucumber green mottle mosaic virus (CGMMV). In CP amino acid composition, strains MS-1 and VIROG-43M are typical representatives of CGMMV: their CPs have 98-100% homology to CPs of other tobamoviruses of the group. The CP gene has the same nucleotide composition in pathogenic MS-1 and vaccine VIROG-43M, indicating that strain attenuation is not determined by this gene. The CP amino acid sequences of the two Russian strains are fully identical to the CP sequences of two Greek strains, GR-3 and GR-5. However, the nucleotide sequences of their genes differ in 13 bp, testifying to the difference between the Russian and Greek strains.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

禽流感病毒A/Chicken/Guangdong/SS/94(H9N2)HA基因的克隆及序列分析

禽流感是由A型流感病毒引起的禽的一种疾病综合症.1878年首次在意大利爆发流行,当时称该病为"鸡瘟".之后,许多国家和地区都有该病的报道,包括美国、英国、澳大利亚、爱尔兰、比利时、荷兰、法国、俄罗斯、加拿大、以色列、匈牙利、日本、中国(包括香港)等[1-3].     

绵羊肺腺瘤病毒中国NM株部分gag基因的克隆与序列分析

参照GenBank中已发表的绵羊肺腺瘤病毒的基因序列,设计合成一对引物,对绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)内蒙株的gag基因中主要编码CA蛋白的基因段进行PCR扩增,产物经琼脂糖凝胶电泳分析,呈现一条约897bp的特异条带,将其回收后克隆入pMD—18T载体中,并进行序列测定与分析。结果表明,与南非株(基因序列号NC—001494)的gag基囚序列比较,核苷酸同源性为83％,推导出的氨基酸同源性为84％。与美国株(基因序列号AF105220)的gag基因序列比较,核苷酸同源性为81．5％,氨基酸同源性为83％。这是我国首次报道的绵羊肺腺瘤病毒的gag基因的一段序列,为我国科研工作者进行更深入的研究奠定基础。     

猪Ⅱ型圆环病毒ORF1基因克隆及在E.coli中的表达

猪圆环病毒(porcinecircovirus ,PCV)属圆环病毒科(Circoviridae) ,为单股负链DNA病毒,以滚环方式进行复制,是目前已知的最小的动物病毒之一.PCV有2种基因型即PCV 1和PCV 2 ,两者细胞培养均不引起病变.前者广泛存在于猪源肾细胞中,但并不引起感染猪发病,其基因组为1 75 9bp ;后者首先由Allan等[1 ] 从患断奶猪多系统衰弱综合症(postweaningmultisystemicwastingsyndrome ,PMWS)的猪群中分离到,被证明为PMWS的重要病原.PCV 2主要侵害感染猪的免疫系统[2 ] ,从而诱发猪体的免疫抑制.PCV 2常和呼吸与繁殖障碍综合征病毒(PRRSV)…     

流行性乙型脑炎病毒减毒株SA14-14-2E基因稳定性研究

为研究乙脑病毒减毒株SA14-14-2 E蛋白基因稳定性,将乙脑病毒减毒株SA14-14-2在原代地鼠肾细胞(PHK)上传至18代,应用RT-PCR分别扩增PHK6代、PHK7代、PHK8代、PHK13代、PHK18代E蛋白基因并测序后,与Genebank中乙脑病毒减毒株SA14-14-2(D90195)进行比较分析。PHK6、PHK7、PHK8代病毒与D90195 E蛋白核苷酸和氨基酸序列完全相同。PHK13、PHK18代病毒与D90195E蛋白核苷酸序列同源性分别为99.8%、99.7%,与D90195E蛋白氨基酸序列同源性分别为99.6%、99.4%。各代次病毒E蛋白与减毒相关氨基酸未发生改变,同时所有突变的氨基酸均非SA14原有的,故不是恢复性突变。结果表明乙脑病毒减毒株SA14-14-2的遗传学特性稳定,从分子水平证明乙脑病毒减毒株SA14-14-2及其生产的疫苗具有安全性。     

Cloning and sequence analysis of genome from the Inner Mongolia strain of the endogenous betaretroviruses (enJSRV)

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19- T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame (orf-x) that overlaps the 3' end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain (Accession No. AF153615) and USA JSRV21 strain (Accession No. AF105220). The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.     

Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria

The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.     

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses （enJSRV）

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia （enJSRV-NM）, we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame （orf-x） that overlaps the 3＇ end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain （Accession No.AF153615） and USA JSRV21 strain （Accession No. AF105220）. The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.     

南极嗜冷假单胞菌7197 S- 腺苷同型半胱氨酸水解酶 ( SAHH) 基因的克隆与序列分析

从南极普利兹湾深海沉积物中筛选到一株耐冷菌株7197,其16S rDNA序列分析表明该菌株属于假单胞菌属（Pseudomonas）。作者通过设计引物,从该菌的全基因组DNA中克隆到编码S-腺苷-L-高半胱氨酸（SAHH）的完整ORF,全长为1424bp。使用DNAMAN（5,1）软件对全长ORF为1424bp的SAHH基因进行分析,SAHH基因编码一个由474AA残基组成、分子量预计为52523Da的SAHH蛋白质,与Psychrobacter sp．273—4的SAHH有96．84％的相似性;与Acinetobacter sp．ADP1的SAHH有79％的相似性;与Pseudomonas fluorescens Pf-5的SAHH有75％的相似性。     

嗜盐古生菌AJ4中BR蛋白基因部分片段和16S rRNA基因序列研究

为了研究分析新疆阿尔金山国家自然保护区阿牙克库木湖嗜盐古生菌物种与细菌视紫红质(bacteriorhodopsin ,BR)蛋白资源 ,对分离纯化到的极端嗜盐古生菌AJ4 ,采用PCR方法扩增出其 16SrRNA基因 (16SrDNA)和编码螺旋C至螺旋G的BR蛋白基因片断 ,并测定了基因的核苷酸序列 .通过BR蛋白部分片段序列分析表明 ,BR蛋白中对于完成质子泵功能以及与视黄醛结合的关键性氨基酸残基均为保守序列 ,位于膜内侧的序列比位于膜外侧的序列更保守 ;基于BR蛋白基因和16SrDNA序列的同源性比较以及 16SrDNA序列的系统发育学研究表明 ,AJ4是Haloarcula属中新成员 .由此建立了一种快速筛选具有新BR蛋白的新嗜盐古生菌的方法 .     

Specifics of the coat protein gene in Russian strains of the cucumber green mottle mosaic virus

The primary structure of the coat protein (CP) gene was examined for pathogenic strain MS-1 and vaccine strain VIROG-43M of the cucumber green mottle mosaic virus (CGMMV). In CP amino acid composition, strains MS-1 and VIROG-43M are typical representatives of CGMMV: their CPs have 98–100% homology to CPs of other tobamoviruses of the group. The CP gene has the same nucleotide composition in pathogenic MS-1 and vaccine VIROG-43M, indicating that strain attenuation is not determined by this gene. The CP amino acid sequences of the two Russian strains are fully identical to the CP sequences of two Greek strains, GR-3 and GR-5. However, the nucleotide sequences of their genes differ in 13 bp, testifying to the difference between the Russian and Greek strains.     

Diversity of carbazole-degrading bacteria having the car gene cluster: isolation of a novel gram-positive carbazole-degrading bacterium

Twenty-seven carbazole-utilizing bacterial strains were isolated from environmental samples, and were classified into 14 groups by amplified ribosomal DNA restriction analysis. Southern hybridization analyses showed that 3 and 17 isolates possessed the car gene homologs of Pseudomonas resinovorans CA10 and Sphingomonas sp. strain KA1, respectively. Of the 17 isolates, 2 isolates also have the homolog of the carAa gene of Sphingomonas sp. strain CB3. While the genome of one isolate, a Gram-positive Nocardioides sp. strain IC177, showed no hybridization to any car gene probes, PCR and sequence analyses indicated that strain IC177 had tandemly linked carAa and carC gene homologs whose deduced amino acid sequences showed 51% and 36% identities with those of strain KA1.     

Saprospira species—Natural predators

SomeSaprospira spp. from fresh waters have been shown to be predatory on other aquatic bacteria, particularly gliding bacteria. Some can be grown as well axenically but others so far have been cultured only on the host bacteria for which they show a degree of specificity. A marine strain resemblingSaprospira grandis, although capable of growth axenically, is also a predator on a marineCytophaga sp.S. grandis ATCC 23119 failed to grow on the base media or media overgrown with host cells of otherSaprospira spp.